The hedgehog (hh) signaling pathway is implicated in patterning both invertebrate and vertebrate embryos. Genetic analysis in Drosophila established that the cubitus interruptus (ci) is the most downstream component of the signaling pathway. There are three ci homologs in vertebrate, Gli1, Gli2 and Gli3. The overall goal of the proposal is to determine the distinct and yet overlapping function of these three Gli genes in hh signaling pathway by using the knock-in approach. Specifically, the endogenous coding sequence of Gli2 will be replaced by that of Gli1 and Gli3. This project will be done in three steps. First, the 5' region of Gli2 genomic region will be analyzed to select a knock-in site. In the meantime, Gli1 and Gli3 knock-in construct will be made and tested in Sonic hedgehog responsive cell line MNS70. In the second step, knock-in constructs will be introduced into embryonic stem cells. Chimeric embryos and germline mutant mice will be generated. Finally, the phenotypes associated with heterozygous and homozygous mutant mice will be analyzed by using various marker genes in the spinal cord, lung and skeleton. Studies on the function of Gli genes have direct relevance to human development and disease since homolog of these three genes exist in humans and mutations in these genes have been implicated in cancers and developmental defects.